Orthoester compositions for affinity purification of oligonucleotides

Abstract

Compounds and methods for purifying oligonucleotides such as RNA and DNA. A target oligonucleotide is reacted with an orthoester linker comprising an affinity tag to form an orthoester oligonucleotide-orthoester linker conjugate which is subjected to a purification technique to separate the target oligonucleotide from impurities such as truncated oligonucleotides. The orthoester linker can be then removed under mild conditions to generate the target oligonucleotide in high purity.

We claim: 
     
         1 - 24 . (canceled) 
     
     
         33 . An orthoester linker of formula (Id)
                       wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  is C 1 -C 24  alkyl, C 1 -C 24  heteroalkyl, C 2 -C 24  alkenyl, C 2 -C 24  heteroalkenyl, C 2 -C 24  alkynyl, C 2 -C 24  heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, any substituted equivalent or any combination thereof provided that the total number of carbons doesn’t exceed 24 and the remaining R 1 , R 2 , R 3 , R 4 , R 5 , R 6  and R 7  are H, C 1 -C 6  alkyl, C 2 -C 6  alkenyl, C 2 -C 6  alkynyl, halo- or hetero-substituted equivalents, C 3 -C 12  carbocyclyl or heterocyclyl and substituted equivalents with the proviso that two R groups of R 1  to R 7  groups do not form a fused ring with the cyclic orthoester as shown below:
                     
   X is F, Cl, Br, or a mono-,bis or tris-halosubsituted methyl; and n is 0 or 1, wherein at least one of R 1 , R 2 , R 3 , R 4 , R 5 , R 6  or R 7  comprises an affinity tag.   
     
     
         34 . The orthoester linker of  claim 33 , wherein the affinity tag is a fluorous tag or a hydrophobic tag with a cLogP value of at least 3. 
     
     
         35 . The orthoester linker of  claim 33 , wherein n is 0 and at least two of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are each independently C 1 -C 24  alkyl, C 1 -C 24  heteroalkyl, C 2 -C 24  alkenyl, C 2 -C 24  heteroalkenyl, C 2 -C 24  alkynyl, C 2 -C 24  heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl or any substituted equivalent, and all the remaining R groups of R 1 , R 2 , R 3 , R 4 , R 5 , and R 6  are H, provided that the two R groups that are not H are not linked together to form a fused ring with the cyclic orthoester. 
     
     
         36 . The orthoester linker of  claim 33 , wherein one of R 1 , R 2 , R 3 , R 4 , R 5  or R 6  is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted aryl. 
     
     
         37 . The orthoester linker of  claim 33  having the structure:
                     
 wherein R 11  is H, C 1 -C 23  alkyl, C 1 -C 23  heteroalkyl, C 1 -C 23  substituted alkyl, C 2 -C 23  alkenyl, C 2 -C 23  heteroalkenyl, C 2 -C 23  substituted alkenyl, C 2 -C 23  alkynyl, C 2 -C 23  heteroalkynyl, C 2 -C 23  substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl; R 7  is H or CH 3 . Z is CR a R b , O, S, NR a , NR a CO, CONR a , wherein R a  and R b  are each independently H, C 1 -C 6  alkyl, or R a  and R 11  together form a heterocycle with N; X is F, Cl, Br, or a mono-,bis- or tris-halosubstituted methyl; and n is 0 or 1, R 11  comprises an affinity tag. 
 
     
     
         38 . The orthoester linker of  claim 33  having the structure:
                     
 wherein R 11  and R 12  are each independently H, C 1 -C 23  alkyl, C 1 -C 23  heteroalkyl, C 1 -C 23  substituted alkyl, C 2 -C 23  alkenyl, C 2 -C 23  heteroalkenyl, C 2 -C 23  substituted alkenyl, C 2 -C 23  alkynyl, C 2 -C 23  heteroalkynyl, C 2 -C 23  substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl provided that at least one of R 11  and R 12  comprises an affinity tag; R 7  is H or CH 3 . Z are each independently CR a R b , O, S, NR a CO, CONR a  or NR a  wherein R a  and R b  are each independently H, C 1 -C 6  alkyl, or R a  and R 11  or R a  and R 12  together form a heterocycle with N; X is F, Cl, Br, or a mono-,bis or tris-halosubstituted methyl; and n is 0 or 1. 
 
     
     
         39 . The orthoester of  claim 37  wherein n is 0 and Z is O. 
     
     
         40 . A fluorous orthoester linker having the structure:
                       Wherein Z is CH 2 . O, S, NR a , or NR a CO wherein R a  is H or C 1 -C 6  alkyl; R 7  is H, methyl, ethyl. n-propyl, phenyl or benzyl; X is H, F, Cl, Br, or a mono-,bis- or tris-halosubstituted methyl or cyano-; m is an integer from 0 to 12; n is 1 or 2.   
     
     
         41 . An orthoester linker having the structure selected from:
                                                                                                               .   
     
     
         42 . An orthoester linker of formula (IIIa):
                       wherein X 1  is H, F, an azido, a protected sulfhydryl, a protected poly-sulfhydryl, a poly-histidine, a protected amino group, a protected hydrazide group, a protected oxyamine group, a cyclooctyne, a conjugated diene, a C 2  alkenyl group, a C 2  substituted alkenyl group, a C 2  alkynyl group or a C 2  substituted alkynyl group; R 18  and R 19  are each independently H, F, C 1-3  heteroalkyl or C 1-3  substituted alkyl; X 2  is H, CH 3 , F, Cl, Br, or a mono-,bis- or tris-halosubstituted methyl or cyano-; n is 1 or 2; and m is an integer ranging from 0 to 12.   
     
     
         43 . The orthoester linker of  claim 42 , wherein the orthoester linker is:
                       .